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L‑Lactate Dehydrogenase B May Be a Predictive Marker for Sensitivity to Anti‑EGFR Monoclonal Antibodies in Colorectal Cancer Cell Lines

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L‑Lactate Dehydrogenase B May Be a Predictive Marker for Sensitivity to Anti‑EGFR Monoclonal Antibodies in Colorectal Cancer Cell Lines 4710 ONCOLOGY LETTERS 17: 4710-4716, 2019 L‑Lactate dehydrogenase B may be a predictive marker for sensitivity to anti‑EGFR monoclonal antibodies in colorectal cancer cell lines AYUMU NAGAMINE1,2, TAKUYA ARAKI1,2, DAISUKE NAGANO1, MITSUE MIYAZAKI3 and KOUJIROU YAMAMOTO1,2 1Department of Clinical Pharmacology and Therapeutics, Gunma University Graduate School of Medicine; 2Department of Pharmacy, Gunma University Hospital; 3Division of Endocrinology Metabolism and Signal Research, Gunma University Initiative for Advanced Research and Institute for Molecular and Cellular Regulation, Maebashi, Gunma 371‑8511, Japan Received October 15, 2018; Accepted January 31, 2019 DOI: 10.3892/ol.2019.10075 Abstract. Recently, proteins derived from cancer cells have Introduction been widely investigated as biomarkers for predicting the efficacy of chemotherapy. In this study, to identify a sensitive Various factors such as genetic variations and changes in biomarker for the efficacy of anti‑epidermal growth factor mRNA expression patterns are known to influence drug effi- receptor monoclonal antibodies (anti-EGFR mAbs), proteins cacy, and such factors have been studied extensively. However, derived from 6 colorectal cancer (CRC) cell lines with drug responses are affected by changes in the conformation, different sensitivities to cetuximab, an anti‑EGFR mAb, were localization, and expression of numerous proteins, which analyzed. Cytoplasmic and membrane proteins extracted from are regulated by mutations and mRNA expression levels. In each CRC cell line were digested using trypsin and analyzed 2014, Zhang et al reported that only 32% of the genes showed comprehensively using mass spectrometry. As a result, 148 and statistically significant positive mRNA-protein correlation 146 peaks from cytoplasmic proteins and 363 and 267 peaks in 86 CRC samples (1). Therefore, proteomics analysis‑the from membrane proteins were extracted as specific peaks for direct evaluation of the expression levels and modifications of cetuximab‑resistant and ‑sensitive CRC cell lines, respectively. proteins‑has been focused on recently as a powerful explora- By analyzing the proteins identified from the peptide peaks, tion method for identifying predictive biomarkers for the cytoplasmic L-lactate dehydrogenase B (LDHB) was detected efficacy of chemotherapeutic drugs. as a marker of cetuximab sensitivity, and it was confirmed The expression levels of some serum proteins have been that LDHB expression was increased in cetuximab‑resistant reported to be useful indicators of sensitivity to chemotherapy CRC cell lines. Furthermore, LDHB expression levels were for cancer, and Li et al reported that variation in serum LDH significantly upregulated with the acquisition of resistance to level was useful as a predictive biomarker of efficacy of beva- cetuximab in cetuximab‑sensitive CRC cell lines. In conclu- cizumab in non‑small cell lung cancer (NSCLC) patients (2). sion, LDHB was identified as an important factor affecting Furthermore, proteomic studies analyzing several serum cetuximab sensitivity using comprehensive proteome analysis proteins using matrix‑assisted laser desorption/ionization for the first time. mass spectrometry (VeriStrat; Biodesix, Boulder, CO), clas- sified NSCLC patients treated with erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, into two groups with good or poor prognosis (3-10). However, serum proteins derived from tumor tissue and circulating in blood stably represent only a small fraction of total protein Correspondence to: Dr Takuya Araki, Department of Clinical derived from a tumor. Thus, to find more suitable predictive Pharmacology and Therapeutics, Gunma University Graduate School biomarkers for sensitivity to anticancer drugs, the analysis of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma 371‑8511, should include not only proteins released into blood but all Japan E‑mail: tkyaraki@gunma‑u.ac.jp proteins derived from a tumor. In 2015, Sun et al (11) reported that a high level of L-lactate Key words: L-lactate dehydrogenase B, anti-epidermal growth dehydrogenase B (LDHB) expression in tumor tissue was factor receptor monoclonal antibodies, colorectal cancer, effect associated with poor overall survival in oral cancer patients predictor, proteome treated with paclitaxel, and Ferrer et al (12) also reported that 17 kDa membrane‑associated protein expression in tumor tissue could predict sensitivity to platinum‑based therapy, EGFR inhibitors, and the proteasome inhibitor bortezomib in NAGAMINE et al: LDHB AS A PREDICTIVE MARKER FOR SENSITIVITY TO ANTI‑EGFR MONOCLONAL ANTIBODIES 4711 lung adenocarcinoma in 2018. In a meta‑analysis of clinical (Salisbury, UK). The COLO320DM cell line was purchased studies, Li et al (13) showed that aldehyde dehydrogenase 1 from the Japanese Collection of Research Bioresources could be a predictor of response to neoadjuvant chemotherapy Cell Bank (Osaka, Japan). The SW48 cell line was obtained in breast cancer. Furthermore, in recent years, proteins in from the American Type Culture Collection (Manassas, tumor cells have been comprehensively analyzed. Yu et al (14) VA, USA). These cell lines were classified into three groups identified some predictive marker proteins for sensitivity to according to cetuximab sensitivity: C99 and SW48 in the platinum‑containing drugs in patients with ovarian cancer. cetuximab‑sensitive group (Cmab‑S), C10 and HT55 in the Moreover, Chauvin et al (15) detected predictive marker cetuximab‑partial resistance group (Cmab‑PR), and CACO2 proteins for the efficacy of 5‑fluorouracil (5‑FU) in patients and COLO320DM in the cetuximab‑resistance group with locally advanced rectal cancer. As mentioned above, the (Cmab‑R). Reagents for culture [culture medium, fetal bovine usefulness of proteome analysis of tumor tissues to develop serum (FBS), and supplements] and sample preparation predictive markers for the efficacy of certain small molecule (dithiothreitol, iodoacetamide, and trypsin) were purchased drugs has been demonstrated. On the other hand, although from Wako Pure Chemical Industries (Osaka, Japan). All other L‑lactate dehydrogenase A (LDHA) expression levels in reagents were obtained from commercial sources, and those tumors have been reported to correlate with cetuximab sensi- used to analyze peptides were graded for high‑performance tivity in patients with Ewing's sarcoma (16) and gankyrin has liquid chromatography, liquid chromatography‑tandem mass been reported to contribute to resistance to chemotherapy spectrometry (LC-MS/MS), or analytical use. Standard containing bevacizumab in CRC (17), comprehensive proteome peptides used for MS analysis were synthesized by Eurofins analysis to identify predictive biomarkers for the efficacy of Genomics Japan (Tokyo, Japan). antibody drugs has not been conducted. Anti-EGFR monoclonal antibodies (anti-EGFR mAbs), Cell culture and sample preparation. Six CRC cell lines with including cetuximab and panitumumab, are key drugs in the different sensitivities to cetuximab were used in this study treatment of colorectal cancer (CRC) and are highly effective (Table I) (29). CACO2, COLO320DM, C10, HT55, and C99 cells for some CRC patients. On the other hand, anti‑EGFR mAbs were cultured in a humidified incubator at 37˚C in the presence are very expensive, and are also known to cause serious adverse of 5% CO2, while SW48 cells were cultured in a 37˚C incubator effects such as an infusion reaction or skin rash. Therefore, with no supplemental CO2. Cetuximab‑acquired resistance these drugs should be used only for patients in which an effec- cell lines (SW48R and C99R) were generated upon continuous tive response is expected. Many clinical trials have concluded exposure of SW48 and C99 cell lines to cetuximab according to that variations in the KRAS gene are a crucial factor affecting the method of Troiani et al (30). Briefly, for a period of 8 months, the clinical efficacy of anti‑EGFR mAbs. Anti‑EGFR mAbs SW48 and C99 cells were continuously exposed to cetuximab have been recommended to be used for wild‑type KRAS CRC to increase the inhibition of 50% of cancer cell growth (IC50), patients, approximately 60% of all CRC patients. However, and the final concentration was 12.8 µg/ml. Cytoplasmic and more than 50% of patients with wild‑type KRAS tumors do not membrane proteins were extracted from 80% confluent cell receive a therapeutic benefit from anti‑EGFR mAbs (18-22). lines using a Minute Plasma Membrane Protein Isolation kit Even when variations of NRAS, BRAF, and PIK3CA mutations (Invent Biotechnologies, Inc., Plymouth, MN, USA), and the or EGFR overexpression are taken into account, the primary concentrations of the extracted proteins were measured using cause of resistance to anti‑EGFR mAbs in more than half a DC™ Protein assay kit (Bio‑Rad Laboratories, Inc., Hercules, of wild-type KRAS CRC patients with poor drug responses CA, USA). Cytoplasmic and membrane protein extracts were remains unknown (20,23-28). As mentioned above, although diluted to 2.0 and 1.0 mg/ml, respectively, and assayed immedi- genome research is widely performed and has many advan- ately or stored at ‑80˚C until assay. tages such as the ease of the procedure, it has become clear that fluctuation in protein expression, which directly affects Mass spectrometry analysis. As a pretreatment for cytoplasmic drug efficacy, cannot be explained with genome information and membrane protein samples, protein samples (45 µl) were alone. Thus, factors affecting
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